ATG16L1 was recently identified in genome-wide association studies as a Crohn's disease susceptibility gene, thereby implicating autophagy in the pathogenesis of Crohn's disease. While the strongest genetic association results in a threonine to alanine transition at codon 300 (T300A) that may alter function of the resultant protein and initial studies have not yet identified Crohn's disease-associated changes in ATG16L1 expression, studies using hypomorphic mice expressing low ATG16L1 levels indicate that the reduced expression of ATG16L1 may significantly alter its function in intestinal epithelial cells. Overall, there is a lack of knowledge of how the expression of ATG16L1 and other autophagy genes are regulated. MicroRNAs are 21-24 nucleotide noncoding RNA molecules that negatively regulate gene expression by binding to complementary sequences in the 3'untranslated region (3'UTR) of mRNA transcripts and directing either mRNA degradation or protein translational inhibition. We have previously identified microRNAs that are differentially-expressed in Crohn's disease and ulcerative colitis tissues. An in silico analysis found that the ATG16L1 3'UTR contains over 25 putative microRNA binding sites and 3 naturally occurring SNPs that map to 3 distinct putative microRNA binding sites. Our preliminary results indicate that the ATG16L1 3'UTR plays a significant role in regulating ATG16L1 expression. Our long-term goal is to determine the role of microRNAs in the pathogenesis of inflammatory bowel diseases, such as Crohn's disease. The objective here is to determine the influence of 3'UTR microRNA binding sites in the regulation of ATG16L1 expression and function. Our central hypothesis is that microRNA play a significant role in regulating ATG16L1 expression and that microRNA-mediated alterations in ATG16L1 expression significantly impact autophagy in intestinal epithelial cells. We will address this central hypothesis with the following two specific aims: (1) To identify microRNA binding sites regulating ATG16L1 expression and subsequent autophagy and xenophagy; (2) To determine microRNA binding site SNP effects on ATG16L1 expression and subsequent autophagy and xenophagy. These aims will be pursued using established microRNA and autophagy functional assays and generating intestinal epithelial cells harboring the microRNA binding site SNPs using somatic cell gene targeting. Such results are expected to have an important positive impact because the determination of microRNA regulation of ATG16L1 expression and autophagy provides a physiologic mechanism by which autophagy can be regulated at a cellular level and influence disease processes. These results will confirm the utility of somatic cell mutations to study SNP influences on 3'UTR microRNA binding site function and may provide future microRNA-associated avenues by which autophagy-associated diseases may be targeted.